The present invention relates to a method for assessing the sensitivity of an HIV-1 sample to zidovudine and to diagnostic assays for use in such assessment.
One group of viruses which has recently assumed a particular importance are the retroviruses. Retroviruses form a sub-group of RNA viruses which, in order to replicate, must first xe2x80x98reverse transcribexe2x80x99 the RNA of their genome into DNA (xe2x80x98transcriptionxe2x80x99 conventionally describes the synthesis of RNA from DNA). Once in the form of DNA, the viral genome is incorporated into the host cell genome, allowing it to take full advantage of the host cell""s transcription translation machinery for the purposes of replication. Once incorporated, the viral DNA is virtually indistinguishable from the host""s DNA and, in this state, the virus may persist for as long as the cell lives. As it is believed to be invulnerable to attack in this form, any treatment must be directed at another stage of the virus life cycle and would, have to be continued until all virus-infected cells have died.
A species of retrovirus has also been reproducibly isolated from patients with AIDS and is now named as human immunodeficiency virus (HIV-1) and is also known as human T-cell lymphotropic virus III (HTLV III), AIDS associated retrovirus (ARV), or lymphadenopathy associated virus (LAV).
This virus has been shown preferentially to infect and destroy T-cells bearing the OKT4 surface marker and is now generally accepted as the aetiologic agent of AIDS. The patient progressively loses his set of T-cells, upsetting the overall balance of the immune system, reducing his ability to combat other infections and pre-disposing him to opportunistic infections which frequently prove fatal. Thus, the usual cause of death is AIDS victims is by opportunistic infection, such as pneumonia or virally induced cancers, and not as a direct result of HIV infection.
The complete nucleotide sequence of the AIDS virus HIV-1 or as it was previously known HTLV-III has been elucidated (Ratner, L., et al. Nature, Vol. 313, p 277, 24 Jan. 1985).
Recently, HIV-I has also been recovered from other tissue types, including B-cells expressing the T4 marker, macrophages and non-blood associated tissue in the central nervous system. This infection of the central nervous system has been discovered in patients expressing classical AIDS symptoms and is associated with progressive demyelination, leading to wasting and such symptoms as encephalopathy, progressive dysarthria, ataxia and disorientation. Further conditions associated with HIV infection are the asymptomatic carrier state, progressive generalised lymphadenopathy (PGL) and AIDS-related complex (ARC). HIV-1 can also be present in other tissues or physiological fluids such as urine, plasma, blood, serum, semen, tears, saliva or cerebrospinal fluid.
3xe2x80x2-Azido-3xe2x80x2-deoxythymidine-(hereafter called xe2x80x9czidivudinexe2x80x9d) is used in the control of HIV infections including AIDS and ARC. Zidovudine is a thymidine analogue whose triphosphate form inhibits the replication of the human immunodeficiency virus (HIV) by competitive binding to viral reverse transcriptase (RT) and DNA chain termination after phosphorylation by cellular enzymes (Furman, P. A et al., Pric. Natl. Acad. Sci. USA, 1986, 83: 8333). It is an effective antiviral agent both in vitro and in vivo against a variety of retroviruses (Mitsuya H et al., Cancer Res., 1987, 47: 3140; Ruprecht R. M et al., Nature, 1986, 323: 476.) and has been demonstrated to improve the quality and length of life of patients with AIDS and advanced ARC (Fischl M. A et al., N. Engl. J. Med., 1987, 317: 185; Schmitt F. A. et al, N. Engl. J. Med 1988, 31 9: 1573; Greagh-Kirk T et al, J. Am. Med. Assoc., 1988, 260.: 3009) and also in asymptomatics with low CD4+ cell
As with any anti-infective agent, concern about the potential development of resistance has engendered extensive investigations evaluating factors which might potentially alter the sensitivity of retroviruses to zidovudine.
A study carried out to measure zidovudine sensitivity of HIV isolates from patients with AIDS or ARC after zidovudine treatment has in fact revealed that a number of isolates from patients treated for six months or more showed reduced sensitivity to zidovudine whereas isolates from untreated individuals and those treated for less than six months showed uniform sensitivity to the drug (Larder, B. A., Darby, G, and Richman, D. D., Science, Vol. 243, 1731, 31st Mar. 1989).
At present the way to determine the sensitivity of HIV-1 strains to xe2x80x9czidivudinexe2x80x9d) is to isolate HIV-1 from a patient""s peripheral blood lymphocytes. HIV-1 isolates can be made by co-cultivation of peripheral blood lymphocytes (PBL""s) with cells of the continuous line MT-2. (Harada, S., Koyanagt, Y., Yamamoto, N., Science 229, 563 (1985). This procedure can take anything from between four sad fourteen days before HIV-1 can be isolated. The diagnosis of resistant strains of HIV-1 relies firstly on the isolation of virus and then on sensitivity testing by a tissue culture method and so is consequently extremely slow.
The present inventors have discovered the basis for resistance of HIV-1 to zidovutne at the nucleic acid level. Five nucleotide substitutions have been identified in the HIV-1 genome which result in a change of four amino acids in the RT protein. This discovery has important implications for the detection of resistant HIV isolates because of the highly conserved nature of these nucleotide mutations in RT conferring resistance to zidovudine and opens the way to the routine detection of such resistant isolates.
It is possible to carry out a diagnostic assay for the screening of bodily samples from patients for an assessment of the sensitivity of HIV-I to zidovudine. Using the knowledge of the mutations identified as important in the development of highly resistant strains of HIV-1 such as assay can be developed.
An analysis of a group of resistant mutants was carried out by nucleotide sequencing which allowed the identification of mutations in the HIV RT gene that confer resistance to zidovudine. The complete RT coding region (1.7 kb) was obtained far each isolate using polymerase chain reaction (PCR) amplification of infected cell DNA. The nucleotide changes at the five positions in HIV-1 RT that confer resistance to zidovudine are illustrated in FIG. 1. Numbering of the nucleotides of the HIV-1 RT gene is as reported by Ratner et al (Ratner et al., Nature. 313, 277, (1985)).
It is demonstrated that these specific mutations confer zidovudine-resistance, as an infectious molecular HIV clone containing only these nucleotide changes is resistant to zidovudine. (See Example 4).
From analysis of clinical samples it appears that the sensitivity of HIV-1 to zidovudtne changes over a period of time. It appears that mutations may occur at any of one or more of the five identified sites as the time from the onset of treatment with zidovudtne advances and it is clear than as HIV-1 sample which carries all five mutations is highly resistant to zidovudine.
At this time it is not possible to predict any order of occurence of these mutations, although particular attention is being focussed on the two nucleotides of the wild-type DNA sequence (or its corresponding RNA) or to the two nucleotides of the mutant DNA sequence (or its corresponding RNA) set forth in FIG. 1 at the 2772- and 2773- positions.
Accordingly in a fast aspect of the invention there is provided a method for assessing the sensitivity of an HIV-1 sample to zidovudine, which comprises:
(i) isolating nucleic acid from the sample,
(ii) hybridising an oligonucleotide to the nucleic acid, the oligonucleotide being complementary to a region of the wild-type DNA sequence (or its corresponding RNA) or to a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) and terminating at the 3xe2x80x2-end with the nucleotide in the 2328-, 2338-, 2772-, 2773- or 2784-position,
(iii) attempting polymerization of the nucleic acid from the 3xe2x80x2-end of the oligonucleotide,
(iv) ascertaining whether or not an oligonucleotide primer extended product is present.
It is possible to use genomic DNA or RNA isolated from HIV-1 samples in this methodology. Suitable cells for supporting the growth of HIV-1 such as MT-2 cells, are firstly infected with as HIV-1 isolate and incubated for a period of time. The cells are recovered by centrifugation. DNA can thaw be isolated by digestion of the cells with proteinase K in the presence of EDTA and a detergent such as SDS, followed by extraction with phenol (see Example 1 for the methodology used by the inventors for the isolation of HIV-1 DNA).
Well-known extraction and purification procedures are available for the isolation of RNA from a sample. RNA can be isolated using the following methodology. Suitable cells are again infected and incubated for a period of time. The cells are recovered by centrifugation. The cells are resuspended in an RNA extraction buffer followed by digestion using a proteinase digestion buffer and digestion with proteinase K. Proteins are removed in the presence of a phenol/chloroform mixture. RNA can than be recovered following further centriguation steps. (Maniatis, T., et al, Molecular Coning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, (1989)).
Although it is possible to use unamplified nucleic acid, due to the relative scarcity of nucleic acid in an HIV-1 sample it is preferable to amplify it. Nucleic acid may be selectively amplified using the technique of polymerase chain reaction (PCR), which is an in vitro method for producing large amounts of a specific nucleic acid fragment of defined length and sequence from small amounts of a template.
The PCR is comprised of standard reactants using Mg2+ concentration, oligonucleotide primers and temperature Cycling conditions for amplification of the RT Gene using the primers. The primers are chosen such that they will amplify the entire RT gene or a selected Sequence which incorporates nucleotides corresponding to a region of the wild-type DNA sequence of HIV-1 between the nucleotides at the 2328- and 2784- positions set forth in FIG. 1. Example 2 provides a description of PCR used to amplify target nucleic acid.
RNA cannot be amplified directly by PCR. Its corresponding cDNA must be first of all synthesised. Synthesis of cDNA is normally carried out by primed reverse transcription using oligo-dT primers. Advantageously, primers are chosen such that they will amplify the nucleic acid sequence for RT or a selected sequence which incorporates nucleotides corresponding to the region of RNA corresponding to the wild-type DNA sequence or to the region of the mutant DNA sequence set forth in FIG. 1 between the nucleotides 2328- and 2784-. This could be achieved by preparing an oligonucleotide primer which is complementary to a region of the RNA strand which is up-stream of the corresponding sequence of the wild-type DNA sequence between nucleotides 2328- and 2784-. cDNA prepared by this methodology (see Maniatis, T., et al., supra.) can then be used in the saint way as for the DNA already discussed.
The next stage of the methodology is to hybridise to the nucleic acid an oligonucleotide which is complementary to a region of the wild-type DNA sequence (or its corresponding RNA) or to a region of the mutant DNA sequence (or its corresponding RNA) set forth in FIG. 1 and terminating at the 3xe2x80x2-end with the nucleotide at the 2328-, 2338-, 2772-, 2773- or 2784- position.
Conditions and reagents are then provided to permit polymerisation of the nucleic acid from the 3xe2x80x2-end of the oligonucleotide primer. Such polymerisation reactions are well-known in the art.
If the oligonucleotide primer has at its 3xe2x80x2-end a nucleotide which is complementary to a mutant genotype, that is a genotype which has a nucleotide change at the 2328-, 2338-, 2772-, 2773- or 2784- position as set forth in FIG. 1 then polymerization of the nucleic acid sequence will only occur if the nucleic acid of the sample is the same as the mutant genotype. Polymerisation of a wild type nucleic acid sequence will not occur or at least not to a significant extant because of a mis-match of nucleotides at the 3xe2x80x2-end of the oligonucleotide primer and the nucleic acid sequence of the sample.
If the oligonucleotide primer has at its 3xe2x80x2-end a nucleotide which is complementary to the wild-type genotype, that is a genotype which has the wild-type nucleotide at the corresponding 2328-, 2338-, 2772-, 2773- or 2784- position as set forth in FIG. 1, then these will be polymerisation of a nucleic acid sequence which is wild-type at that position. There will be no polymerisation of a nucleic acid which has a mutant nucleotide at the 3xe2x80x2-position.
The preferred length of each oligonucleotide is 15-20 nucleotides. The oligonucleotide can be prepared according to methodology well known to the man skilled in the art (Koster, H., Drug Research, 30 p548 (1980); Koster, H., Tetrahedron Letters p1527 (1972); Caruthers, Tetrahedron Letters, p719, (1980); Tetrahedron Letters, p1859, (1981); Tetrahedron Letters 24, p.245, (1983); Gate. M., Nucleic Acid Research, 8, p1081, (1980)) and is generally prepared using an automated DNA synthesiser such as an Applied Biosystems 381A synthesiser.
It is convenient to determine the presence of an oligonucleotide primer extended product. The means for carrying out the detection is by using an appropriate label.
The label may be conveniently attached to the oligonucleotide primer or to some other molecule which will bind the primer extended polymerisation product.
The label may be for example an enzyme, radioisotope or fluorochrome. A preferred label may be biotin which could be subsequently detected using streptavidin conjugated to an enzyme such as peroxidase or alkaline phosphatase. The presence of an oligonucleotide primer extended polymerisation product can be detected for example by running the polymerisation reaction on an agarose gel and observing a specific DNA fragment labelled with ethidium bromide, or Southern blotted and autoradiographed to detect the presence or absence of bawds corresponding to polymerised product. If a predominant band is present which corresponds only to the labelled oligonucleotide then this indicates that polymerisation has not occurred. If bands are present of the correct predicted size, this would indicate that polymerisation has occurred.
For example, DNA isolated from patients"" lymphocytes as described herein is used as a template for PCR amplification using synthetic oligonucleotide primers which either match or mis-match with the amplified sequences. The feasibility of PCR in detecting such mutations has already been demonstrated. PCR using the Amplification Refractory Mutation system (xe2x80x9cARMSxe2x80x9d) (Newton, C. R., et al. Nucleic Acids Research, 17, p.2503; (1989)) Synthetic oligonucleotides are produced that anneal to the regions adjacent to and including the specific mutations such that the 3xe2x80x2 end of the oligonucleotide either matches or mismatches with a mutant or wild-type sequence. PCR is carried out which results in the identification of a DNA fragment (using gel electrophoresis) where a match has occurred or no fragment where a mismatch occurred.
For example, using the 2 oligonucleotides below as PCR primers:
5xe2x80x2-ATG TTT TTT GTC TGG TGT GGT-3xe2x80x2- (1) OR
5xe2x80x2-ATG TTT TTT GTC TGG TGT GAA-3xe2x80x2- (2) plus the common oligonucleotide primer:
xe2x80x9cBxe2x80x9d-5xe2x80x2- GGA TGG AAA GGA TCA CC-3xe2x80x2 it is possible to distinguish between sensitive and resistant virus. DNA is extracted from HIV-1 infected T-cells as described herein and subjected to xe2x80x9cARMSxe2x80x9d PCR analysis using these primers. If the virus is sensitive a 210 bp fragment is generated with oligonucleotide B+(1) but not with B+(2). BY contrast, if the virus is zidovudine resistant a 210 bp fragment is generated with B+(2) but not with B+(1).
The presence of a fragment is identified by using an oligonucleotide primer as described above, i.e. by attempting polymerisation using an oligonucleotide primer which may be labelled for the amplified DNA fragment under stringent conditions which only allow hybridisation of complementary DNA (the only difference is that differential hybridisation does not have to be performed as fragments of DNA amplified by the xe2x80x9cARMSxe2x80x9d method will be the same whether derived from mutant or wild-type DNA, so a common oligonucleotide can be used to detect the presence of these fragments. The sequence of such an oligonucleotide is derived from a DNA sequence within the DNA fragment that is conserved amongst HIV-1 strains).
The above PCR assay may be adapted to enable direct detection of mutations associated with zidovudine resistance is DNA from PBL samples from infected individuals that have not been cultured to obtain virus. As this material generally contains considerably less HIV-1 DNA than that in infected lymphoid cultures a xe2x80x9cdouble PCRxe2x80x9d (or nested set) protocol can be used (Simmonds, P., Balfe, P, Peutherer, J. F., Ludlam, C. A., Bishop, J. O. and Leigh Brown, A. J., J. Virol., 64, 864-872, (1990)) to boost the amount of target HIV-1 RT DNA signal in the samples. The double PCR overcomes the problem of limited amplification of a rare template sequence. Initially a fragment may be amplified from within the RT region which encompasses all the commonly observed mutations. A small amount of the pre-amplified material may be used in the second PCR with primer pans designed to allow discrimination of wild type and mutant residues.
A suitable test kit for use in an assay to determine the resistance status of an HIV-1 sample to zidovudine which makes use of a methodology according to the first aspect of the invention, comprises an oligonucleotide being complementary to a region of the wild-type DNA sequence (or its corresponding RNA) or to a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) and terminating at the 3xe2x80x2-end with the nucleotide in the 2328-, 2338-, 2772-, 2773- or 2784- position, other materials required for polymerisation of the nucleic acid from the 3xe2x80x2- end of the oligonucleotide and means for determining the presence of an oligonucleotide primer extended product. Such other materials include appropriate enzymes, buffer and washing solutions, and a label and a substrate for the label if necessary. If PCR is used to amplify nucleic acid then additional materials such as appropriate oligonucleotide primers which will amplify a region of the wild-type DNA sequence (or its corresponding RNA) or a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) containing one or more of the nucleotides at the 2328-, 2338-, 2772-, 2773- or 2784- position, appropriate enzymes and dNTP""s should be included.
In a second aspect of the invention there is provided a method for determining the sensitivity of as HIV-1 sample to zidovudine which comprises:
(i) isolating the nucleic acid from the sample,
(ii) hybridising the nucleic acid with an oligonucleotide being complementary to a region of the wild-type DNA sequence (or its corresponding RNA) or to a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) containing one or more of the nucleotides at the corresponding 2328-, 2338-, 2772-, 2773- and/or 2784- position,
(iii) ascertaining whether or not any of the resulting hybrids of the oligonucleotide and nucleic acid have complementary nucleotides at one of these positions.
Preferably the oligonucleotide is so designed to form a perfectly matched hybrid with its complement.
Nucleic acid (DNA or RNA) is isolated from a sample by the aforementioned methods as described for the first aspect of the invention.
Similarly, PCR may be used to amplify the RT DNA (or its corresponding RNA) or preferably to amplify a region of the RT DNA (or its corresponding RNA; which incorporates DNA (or its corresponding RNA; containing one or more of the nucleotides at the 2328-, 2338-, 2772-, 2775- and/or 2784- position (see Example 2).
In the second stage of this methodology the nucleic acid is then used to hybridise to oligonucleotides complementary to a region of the wild-type DNA sequence (or its corresponding (RNA) or to a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) containing one or more of the nucleotides at the aforementioned positions.
The oligonucleotide may be of any length depending on the number of nucleotide positions of interest which are being examined. If the oligonucleotide is designed to include a nucleotide at only one position of interest then this nucleotide is preferably at or close to the centre position of the oligonucleotide.
For example, referring to FIG. 1, one oligonucleotide probe for detection of the mutation at nucleotide 2328- would be complementary to the mutated RT gene sequence and would include at its nucleotide corresponding to nucleotide 2328- the nucleotide complementary to the mutated 2328- nucleotide. A second oligonucleotide probe for the wild type HIV-1 RT would include at its nucleotide corresponding to nucleotide 2328- the nucleotide complementary to the wild-type 2328- nucleotide. Oligonucleotide probes designed to detect one or more of the mutations referred to in FIG. 1 are used to detect a specific mutation or that there has been a mutation.
In order to ascertain whether or not the oligonucleotide and nucleic acid sequence have formed a matched hybrid, specific hybridisation conditions are set so that a hybrid is only formed when the nucleotide or nucleotides at one or more of the 2328-, 2338-, 2772-, 2773- and 2784- position is or are complementary to the corresponding nucleotide or nucleotides of the oligonucleotide which either permits hybridisation or no hybridisation. It is important to establish for example the temperature of the reaction and the concentration of salt solution before carrying out the hybridisation step to find conditions that are stringent enough to guarantee specificity (Maniatis, T., et al., Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbour Laboratory Press, (1989)). If the oligonucleotide probe has a DNA sequence which is complementary to a wild-type nucleic acid sequence at one or more of its nucleotides corresponding to the 2328-, 2338-, 2772-, 2773- or 2784- position of the DNA sequence of FIG. 1 then this oligonucleotide will hybridise perfectly to wild-type nucleic acid. If there is no hybridisation then this would suggest that the nucleic acid isolated from the sample contains one or more mutations.
If the oligonucleotide probe has a DNA sequence which is complementary to a mutant nucleic acid sequence at one or more of its nucleotides corresponding to the 2328-, 2338-, 2772-, 2773- or 2784- positions of the DNA sequence of FIG. 1 then this oligonucleotide will hybridise perfectly to mutant nucleic acid. If there is no hybridisation this would suggest that the nucleic acid isolated from the sample contains no such mutation or mutations. The oligonucleotide probes may be labelled as a means of detection as for the fast aspect of the invention.
The hybridisation and subsequent removal of non-hybridised nucleic acids are performed under stringent conditions which only allow hybridisation of the complementary DNA and not the oligonucleotide containing a mismatch (i.e, oligonucleotide specific hybridisation as described for the detection of sickle cell mutation using the xcex2-globin or HLA-DQxcex1 gene (Saikt, R. K., et al., Nature, 324, p163, (1986)), the activated Ras gene (Ver Laan-de, Vries, M., et al., Gene, 50, 313, (1986)) and xcex2-thalassaemia Wonq, C., et al., Nature, 330, p384, (1987)).
The hybridisation may be carried out by immobilisation of the RT nucleic acid sequence onto nitrocellulose, nylon or other solid matrix (eg. dot-blot). It is convenient to determine the presence of an hybrid by using the means of a label. For example, the chemically synthesised oligonucleotide probes can be suitably labelled using enzyme, radiosotope or fluorochrome. A preferred label may be biotin which could be subsequently detected using streptavidin conjugated to an enzyme such as peroxidase or alkaline phosphatase.
Alternatively the hybridisation may be carried out by immobilisation of the chemically synthesised oligonucleotides referred to above, which are unlabelled, onto a solid support referred to above and subsequent hybridisation by a labelled RT nucleic acid sequence as described previously.
In both situations described above for hybridisation suitable control reactions will be incorporated to determine that efficient hybridisation has occurred. (eg the hybridisation of oligonucleotides to a complementary oligonucleotide).
Results would be readily interpreted as the isolated nucleic acid would hybridise to either the wild type oligonucleotide or the mutant oligonucleotide.
A suitable test kit for use in an assay to determine the sensitivity of an HIV-1 sample to zidovudine which makes use of a methodology according to the second aspect of the invention comprises an oligonucleotide being complementary to a region of the wild-type DNA sequence (or its corresponding RNA) or to a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) containing one or more of the nucleotides at the corresponding 2328-, 2338-, 2772-, 2773- and/or 2784- position, other materials required to permit hybridisation. Such materials include appropriate buffers and washing solutions and a label and a substrate for the label if necessary. Normally the oligonucleotide would be labelled. If PCR is used to amplify nucleic acid prior to hybridisation then additional materials such as appropriate oligonucleotide primers which will amplify a region of the wild-type DNA sequence (or its corresponding RNA) or a region of the mutant DNA sequence set forth in FIG. 1 (or its corresponding RNA) containing one or more of the nucleotides at the 2328-, 2338-, 2772-, 2773- or 2784- position, appropriate enzymes and dNTP""s (deoxy nucleotide triphosphates) should be included.
In one alternate format of the assay, the dNTP""s in the amplification may or may not be coupled to a detector molecule such as a radiosotope, biotin, fluorochrome or enzyme.
It is also possible to detect zidovudine resistant mutations is the HIV-1 RT RNA isolated from clinical samples using an RNA amplification system. Using the methodology described by Guatelli et al. (Proc. Natl. Acad. Sci, (USA), 8/7, 1874-1878, (March 1990)) a target nucleic acid sequence can be replicated (amplified) exponentially in vitro under isothermal conditions by using three enzymatic activities essential to retroviral replication: reverse transcriptase, RNase H and a DNA-dependant RNA polymerase. Such a methodology may be employed followed by an hybridisation step to distinguish mutant from wild-type nucleotides as discussed previously.